Enhanced fluorescence readout and reduced inhibition for nucleic acid amplification tests

Abstract

A fluorescent dye and quencher mixture for reporting on nucleic acid amplification from a sample includes a fluorescent intercalating dye, a dye sequestering or quenching agent such as hydroxynapthol blue (HNB) or caffeine, and primers, dNTPs, and a nucleic acid polymerizing enzyme or fragment thereof. The presence of the dye in combination with the dye sequestering or quenching agent improves the overall dynamic range of the fluorescent signal as well as shortens the time needed for visualization or image capture of amplified nucleic acid. The fluorescent dye and quencher mixture also enables the detection of nucleic acids in samples having low copy numbers.

1 . A fluorescent dye and quencher mixture for reporting on nucleic acid amplification from a sample comprising:
 a fluorescent intercalating dye;   hydroxynapthol blue (HNB); and   primers, dNTPs, and a nucleic acid polymerizing enzyme or fragment thereof.   
     
     
         2 . The mixture of  claim 1 , wherein the enzyme comprises polymerase or a fragment thereof. 
     
     
         3 . The mixture of  claim 1 , wherein the enzyme comprises ligase or helicase and polymerase or fragments thereof. 
     
     
         4 . (canceled) 
     
     
         5 . The mixture of  claim 1 , wherein the fluorescent intercalating dye comprises a dimeric fluorescent dye having an emission peak at around 530 nm. 
     
     
         6 . The mixture of  claim 1 , wherein the fluorescent intercalating dye comprises a cyanine dye having an emission peak at around 520 nm. 
     
     
         7 . The mixture of  claim 1 , wherein the fluorescent intercalating dye comprises acridine orange. 
     
     
         8 . A fluorescent dye and quencher mixture for reporting on nucleic acid amplification from a sample using loop-mediated isothermal amplification (LAMP) comprising:
 a fluorescent intercalating dye;   hydroxynapthol blue (HNB); and   LAMP primers, dNTPs, LAMP reaction buffer, and DNA polymerase or a fragment thereof.   
     
     
         9 . The mixture of  claim 8 , wherein the fluorescent intercalating dye comprises a dimeric fluorescent dye having an emission peak at around 530 nm. 
     
     
         10 . The mixture of  claim 8 , wherein the fluorescent intercalating dye comprises a cyanine dye having an emission peak at around 520 nm. 
     
     
         11 . The mixture of  claim 8 , wherein the fluorescent intercalating dye comprises acridine orange. 
     
     
         12 . The mixture of  claim 1 , wherein the sample contains nucleic acid at concentration below 60 copies/μl. 
     
     
         13 . The mixture of  claim 1 , wherein the fluorescent intercalating dye emits a fluorescent signal that is at least 20-50 fold above background following nucleic acid amplification of the sample. 
     
     
         14 . The mixture of  claim 9 , wherein the concentration of the dimeric fluorescent dye is around 5 μM. 
     
     
         15 . A fluorescent dye and quencher mixture for reporting on nucleic acid amplification from a sample comprising:
 a fluorescent intercalating dye;   caffeine; and   primers, dNTPs, and a nucleic acid polymerizing enzyme or fragment thereof.   
     
     
         16 . The mixture of  claim 15 , wherein the mixture contains at least 50 mM caffeine. 
     
     
         17 . A method of using the mixture of  claim 1 , comprising:
 forming a plurality of small volumes from the mixture;   imaging the plurality of small volumes; and   identifying a subset of the plurality of small volumes that emit a positive fluorescent signal.   
     
     
         18 . The method of  claim 17 , wherein the positive fluorescent signal comprises a fluorescent signal that is at or above a pre-defined fluorescent intensity level. 
     
     
         19 . The method of  claim 17 , further comprising counting the number of small volumes from the plurality that emit the positive fluorescent signal. 
     
     
         20 . The method of  claim 17 , further comprising calculating a nucleic acid concentration in the sample based on the number of small volumes that emit a positive fluorescent signal. 
     
     
         21 . The method of  claim 17 , wherein the small volumes comprise microwells, droplets, or emulsions. 
     
     
         22 . (canceled) 
     
     
         23 . A method of improving the fluorescent reporting of a nucleic amplification process that uses a fluorescent intercalating dye, the method comprising:
 providing a sample containing a nucleic acid sequence to be amplified;   adding a mixture containing the fluorescent intercalating dye, hydroxynapthol blue (HNB), dNTPs, primers, and a nucleic acid polymerizing enzyme or fragment thereof.   
     
     
         24 . The method of  claim 23 , wherein the fluorescent intercalating dye and HNB are present together at the beginning of the nucleic acid amplification process. 
     
     
         25 . A fluorescent dye and quencher mixture for reporting on nucleic acid concentration from a sample comprising:
 a sample containing deoxyribonucleic acid (DNA);   a fluorescent intercalating dye; and   hydroxynapthol blue (HNB).